This invention relates to the use of a test to evaluate exposure to mustard gas. The invention provides methods and a kit for use in evaluating exposure and response to mustard.
The use of sulfur mustard, bis-(2-chloroethyl) sulfide (HD) in chemical warfare has been long known. More recently its use in the Iran-Iraq conflict resulted in many deaths and untold suffering. It""s use was a major threat in the Gulf War. Hence, the method of identifying injury due to mustards is an important pursuit for scientists working for the armed forces.
It is believed that nitrogen and sulfur mustard-induced vesication wherein there is separation of the epidermis from the dermis due to the disruption of the connective tissues may be the result of a specific protease. The exact mechanism of its toxicity remains unclear and no effective antidote has yet been reported in the literature. DNA is considered to be its major intracellular target (Papirmeister et al., 1985). Other toxic effects are protease stimulation, cutaneous degradation and blister formation. Smith et al. (1991) and Cowan et al. (1991 ) have demonstrated that HD and another vesicating agent chloroethylethyl sulfide (CEES) stimulate protease activity in NHEK, Hela Cell line and in human peripheral blood lymphocytes. No information, however, exists with respect to the molecular mechanism of mustard-induced protease activation.
Immunohistochemical studies have been done on the protein composition changes in HD-exposed hairless guinea pig skin. Epidermal-dermal junction proteins, namely, bullous pemphigoid antigen, laminin and hemidesmosomal anchoring filament proteins were affected by exposure to HD. More recently, investigators have found that in the mini pig skin, which is more akin to human skin, only one protein in the lamina lucida, thelamini, is affected by HD. These findings strongly suggest that some specific protease(s) may be responsible for HD-induced vesication.
The concept that a specific protease is involved in pathology related to exposure to mustards is important because the use of generalized protease inhibitors could cause serious side-effects. Cowman, et al. have demonstrated that HD and chloroethyl ethyl sulfide (CEES) stimulate protease activity in vitro in human peripheral blood lymphocytes and in vivo in hairless guinea pig skin. However, a definitive characterization of the mustard-stimulated protease is not described in any previous publication.
This invention relates to a protease which can be stimulated by exposure of NHEK cells to mustard in the presence of Ca+ wherein proteolytic activity is inhibited by leupeptin at 1 mM concentration but is not inhibited by pepstatin at 1 mM concentration.
This invention relates to the discovery that toxicity to mustard may be evaluated by diagnostic test means disclosed herein.
Upon electrophoretic separation (sodium dodocyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE))of buffered extract of human skin cells (normal human epidermal keratinocytes (NHEK)) which had been exposed to mustard-type chemical compounds a band at approximately 50,000 to 80,000 daltons molecular weight was found. (The position of the band was determined partly by the % of polyacrylamide gel used, with the band being between 65% and 80% when 12.5% concentration of the gel was used and lower when the 10% gel was used). This new protein was seen in mustard-exposed NHEK, in pig skin, and in skin of hairless mice. The mustard compounds used included chloroethyl ethyl sulfide (CEES) and 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride, also known as nitrogen mustard (HN2). The protein band from NHEK cultures had protease activity that was identical to characteristics of protease stimulated by mustard. Bis-2-chloroethyl sulfide (HD), also known as sulfur mustard, may also be used in similar manner.
The protein band constitutes a biomarker. The marker protein can be used either to raise protective antibodies to protect against the protease or may be used in a kit for identifying presence or absence of the marker in study of tissues taken from individuals who may have been exposed to mustard poisoning. A sequence of 19 amino acids (NH2-GGGAGTTHLNVQWQPSGGV-COOH) from this protein has been identified. Antibodies have been raised against this sequence in rabbits. Using antibodies raised to said protein, it is possible, using means for testing antibody-antigen binding means known in the art such as Western blot, Elisa tests, tagged antibodies, etc., to identify the protein raised to mustard in a test sample. As an example, the protein raised in response to exposure to nitrogen or sulfur mustard was identified by exposing a test sample of protein to antibodies which interact with the sequence -GGGAGTTHLNVQWQPSGGV-(Seq. ID No. 1) and observing whether or not said antibodies bind to a protein in the test sample. In the example, Western blot was used. However, other methods known in the art to identify binding of antibodies to specific proteins, including use of tagged antibodies, are appropriate. Tags for use in the method of the invention include colorometric tags, fluorescing tags, and radioactive tags.
At present, pathological changes resulting from mustard poisoning are identified by histopathologic and electron microscopic means. The instant invention provides more economical and efficient means of identifying mustard-related pathologies. The improved diagnostic tests make it possible to treat such pathologies more expeditiously.
Materials and Methods
An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and characterize protease stimulated by some common mustards, including 1-chlorethyl ethyl sulphide (CEES) and 2-chloro-N-(2 chloroethyl)-N-methylethanamine hydrochloride (HN2). These cells provide an appropriate human non-tumor primary skin cell culture model which makes it possible to perform the experimental manipulations necessary to study the effect of vesicants.
CEES was obtained from Aldrich Chem. Milwaukee, Wis. HN2 was purchased from Merck and Co. West Point, Pa. NHEK stock culture was purchased from Clonetics Corp. San Diego, Calif. Sulfur mustard (HD)-exposed NHEK sample was obtained from USMRICD, APG, MD. The peptide substrate TRY (carbobenzoxy-valyl-glycl-arginine-4-nitraline acetate) was obtained from Boehringer Mannheim Biochemical in Indiana. Protease inhibitors diisopropyl fluoro phosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), leupeptin, E-64 and pepstatin were purchased from Signa Chemical Company in Missouri.
Cell Culture
Secondary cultures of (NHEK) were grown up to 100% plus confluence as described previously (Mol et al., 1989). Cells were exposed to different concentrations of CEES and HN2 at ambient conditions of incubation in a humidified atmosphere of 5% CO2/95% air at 37xc2x0 C. for 24 hours.
It has been possible to identify vesicant-induced proteases. Using the methods of the invention, it is also possible to demonstrate and test protectants against the offending protease.
Chromogenic Peptide Substrate Protease Assay (CPSPA).
This assay was performed using chromozym TRY substrate (2.5 mM) following the method of Friberger (1982) as described by Smith and Cowan (Cell Biol.Toxicol. 7, 238-248 (1991)). The chromogenic peptide, when cleaved by protease, releases p-nitroaniline (pNA) producing a change in absorbance measured spectrophotometrically at 405 nm. The findings obtained by chromogzym (TRY) peptide substrate protease assay (CPSPA) revealed the optimum mustard concentration and time for protease stimulation to be about 200 xcexcM CEES or 100 xcexcM HN2 over about 16 hours.
Using the methods of the invention, it has been possible to detect an tissue injury-related protease. Both colorimetric and electrophoretic assays are disclosed.
The chromogenic peptide substrate protease assay (CPSPA) of Cowan, (1991) was practiced in order to reproduce Cowan""s work on the observation of vesicant-induced protease stimulation by studying the effects of both CEES and HN2 in NHEK. Mustard-induced protease stimulation was found only with the trypsin-specific substrate. This indicated that mustards may stimulate serine protease(s).
To identify mustard-stimulated protease, SDS-polypeptide complexes were separated by SDS-PAGE on 10% to 12.5% polyacrylamide gel. Mustard-treated cells were solubilized in sample buffer and boiled for 5 minutes at 95xc2x0 C. Several protease inhibitors were included during the wash and after solubilization using the protease inhibitor sampler kit, P6548, from Molecular Probes, Eugene, Oreg., U.S.A. following the instructions therewith. Densitometric analysis was done in the form of arbitrary optical density units (Bio-Med Instruments). Western blot analysis was done using rabbit anti-keratine wide spectrum screening primary antibody and peroxidase-conjugated swine immunoglobins to rabbit immunoglobins as secondary antibody. To demonstrate protease activity in the ≅50 to ≈80 kDa band, proteins in the cell extract were separated by Non-SDS-PAGE and then the band was eluted in phosphate buffer at 37xc2x0 C.